A revised mineral nutrient supplement increases biomass and growth rate in Chlamydomonas reinhardtii

Abstract

Interest in exploiting algae as a biofuel source and the role of inorganic nutrient deficiency in inducing triacylglyceride (TAG) accumulation in cells necessitates a strategy to efficiently formulate species-specific culture media that can easily be manipulated. Using the reference organism Chlamydomonas reinhardtii, we tested the hypothesis that modeling trace element supplements after the cellular ionome would result in optimized cell growth. We determined the trace metal content of several commonly used Chlamydomonas strains in various culture conditions and developed a revised trace element solution to parallel these measurements. Comparison of cells growing in the revised supplement versus a traditional trace element solution revealed faster growth rates and higher maximum cell densities with the revised recipe. RNA-seq analysis of cultures growing in the traditional versus revised medium suggest that the variation in transcriptomes was smaller than that found between different wild-type strains grown in traditional Hutner's supplement. Visual observation did not reveal defects in cell motility or mating efficiency in the new supplement. Ni²⁺-inducible expression from the CYC6 promoter remained a useful tool, albeit with an increased requirement for Ni²⁺ because of the introduction of an EDTA buffer system in the revised medium. Other advantages include more facile preparation of trace element stock solutions, a reduction in total chemical use, a more consistent batch-to-batch formulation and long-term stability (tested up to 5 years). Under the new growth regime, we analyzed cells growing under different macro- and micronutrient deficiencies. TAG accumulation in N deficiency is comparable in the new medium. Fe and Zn deficiency also induced TAG accumulation, as suggested by Nile Red staining. This approach can be used to efficiently optimize culture conditions for other algal species to improve growth and to assay cell physiology.

Figure 1. Intracellular micronutrient content

Cells of strains CC-3269 (upright triangles), CC-125 (squares), and CC-1690 (circles), and CC-425 (inverted triangles) Chlamydomonas cells were grown in TAP (filled symbols) and TP (open symbols) media supplemented with Hutner’s mineral nutrient supplement and collected by centrifugation. The cell pellet was washed with 1 mM EDTA to remove extracellular bound metal and dissolved in 30% nitric acid by incubation in 65 °C. The metal content was measured by ICP-MS. The y-axis plots the range of metal content on a per cell basis under the various conditions or for different strains.

Figure 2. Stock solutions are stable over 5 years

The concentrations of Cu (filled squares), Fe (open squares), Mn (filled circles), Se (open circles), and Zn (filled triangles) in stock solutions from a primary iteration of the revised trace element composition were measured by ICP-MS over a period of 61 months. Each solution was stored at room temperature in the dark. Before each sampling, the solution was filtered through a 0.22 μm filter nto a new container. Each metal solution was diluted to an appropriate concentration prior to injection into the ICP-MS.

Figure 3. Supplementation with a revised trace element composition improves cell growth

Strain CC-3269 was grown in TAP medium supplemented with Hutner’s trace elements (light gray) or the revised composition (dark gray). Cell growth was measured by monitoring optical density at 680 nm (A) and by counting cells using a hemocytometer (B). Intracellular Zn (C), Mn (D), Cu (E), and Fe (F) were measured by ICP-MS as described in Figure 1. The average of biological triplicates is shown and is representative of experimental duplicates. Cell counts were performed by two independent researchers for each duplicate without prior knowledge of the source of the supplement.

Figure 4. Pairwise comparison of transcriptomes from cells supplemented with different trace element recipes

A. Logarithmic mean-difference scatterplots of normalized expression fold-change (y axes) vs. mean expression (x axes). The plots were generated from pairwise transcriptome comparisons of wild-type strain 2137 supplemented with Hutner trace element recipe (H₂₁₃₇) (Castruita et al. unpublished), wild-type strain D66 supplemented with Hutner trace element recipe (H_D66) (González-Ballester et al., 2010), and wild type strain 2137 supplemented with an early iteration of the revised trace element mix (mK₂₁₃₇). Cyan lines in scatter plots represent the average fold change. Red and green lines represent the 95^th and 5^th percentile of fold changes, respectively. Gray lines represent a 2-fold difference in fold change. B. Histogram of expression fold-changes for each pairwise comparison grouped into clusters of log₁₀ 0.2.

Figure 5. Optimization of the amount of Ni²⁺ required to induce CYC6 expression in the revised trace elements

RNA was isolated from cells grown in TAP medium supplemented with the revised mineral nutrient mix, a modification of the revised recipe with EDTA reduced so that the molar amount of free species was comparable to the amount in Hutner’s (2.5 μM), or Hutner’s trace elements 5 hours after addition of the indicated amount of NiCl₂. Transcript abundance was measured by RT-PCR and normalized to EIF5A expression. Average C_T value was calculated from technical triplicates. Expression was calculated by the 2^−ΔΔCT method. The average of experimental triplicates is shown.

Figure 6. Comparison of lipid body formation, cell morphology, and culture appearance of different growth conditions

Cells were grown under replete and deficient nutrient regimes as indicated in the figure. Confocal micrographs of Nile Red stained cells in Photomultiplier Tube Trans and Photomultiplier Tube 1 (Nile Red fluorescence) channels are shown in columns 1 and 2, respectively. Chlorophyll autofluorescence is shown in column 3. The overall appearance of the cultures is shown in the column 4. Additionally, cells were imaged on a Zeiss AxioImager Z1 microscope equipped with a color camera (AxioCam HRc) in DIC mode (column 5). Representative cells shown. Scale bars represent 10 μm.

Figure 7. A strategy for deriving species-specific culture media

i.m. = iterative modification of nutrient concentrations to accommodate the cell ionome.